The present invention relates generally to the in vitro growth and differentiation of human epidermal keratinocytes. More particularly, the present invention is concerned with methods and materials for the initiation of primary cultures of human epidermal keratinocyte cells for human tissue, for the storage of these cells in viable frozen condition, for the establishment of secondary cultures recovered from frozen storage which enable serial propagation from the same primary culture, and for the use of these cells in products and procedures for the repair of injury to the skin.
Much of the historic development of cell culture has been based on the growth requirements and responses of fibroblast cells and closely related cell types. Fibroblasts are mesenchymal cells which, with collagen and elastic fibers in an extracellular matrix, compose loose ordinarily areolar connective tissue. Areolar connective tissue sheathes and penetrates muscles, nerves, and glands, and also forms the dermal tissue layer of the skin. Fibroblasts taken from tissues can be routinely cultivated either through many cell generations as karyotypically diploid cells or indefinitely as established cell lines. Thus, in many ways, conventional cell culture technology has been viewed as simulation in vitro of wound healing conditions.
For various reasons, normal epithelial cells and many of the tumors that develop from epithelial cells do not proliferate well in standard media and under conventional culture conditions. Epithelial cells, which cycle continuously in an uninjured body, form the covering tissue for nearly all the free surfaces of the body, including the epidermis of the skin. The basal layer of mammalian epidermis, separated by a basement membrane from the fibroblasts of the underlying dermis, is composed principally of dividing keratinocyte cells in various stages of differentiation. Some keratinocyte cells undergo terminal differentiation (i.e., move outward from the basal layer, increase in size, develop an envelope resistant to detergents and reducing agents, and eventually are shed from the surface). Because conventional tissue culture conditions have strongly favored multiplication of fibroblast-like cells, any epithelial cells that may be in a primary culture inoculum would tend to be overrun by fibroblast cells.
The ability to culture viable layers of nondifferentiated human epidermal keratinocyte cells with minimal fibroblast overgrowth would have considerable application to medical procedures for wound healing. As one example, when human tissue has been severely damaged due to a severe burn, it is necessary to cover the damaged area to reduce fluid loss, prevent infection and reduce scarring. Because autografts are painful and difficult when damage is extensive, other functioning substitutes for skin have been sought, i.e., homografts (skin transplants from live donors or from skin preserved in skin banks), modified skin from animals, synthetic polymeric structures, reconstituted collagen films, and biodegradable synthetic membranes, such as described in Yannas, et al., U.S. Pat. No. 4,060,081.
Many of these skin substitutes have the potential for inducing inflammatory response in a patient caused by rejection of some antigenic substance in the skin substitute by the immune system. Further, it is often the case that a patient with extensive or severe injury suffers considerable risk of infection and death due to immune depression associated with the second and third weeks of a skin graft. Whether an autograft, homograft or skin substitute is employed to cover the damaged tissue, a graft may be unavailing to a patient succumbing to infection unless the skin repair is rapid enough to reduce exposure to the outside environment.
Recent studies have focused on the development of nutritionally optimized and readily defined and reproducible media and culture conditions which may provide a selective advantage to epithelial cells. The ability to culture layers of keratinocyte cells of the skin in vitro from a small inoculum could replace the requirement for autografts in present wound repair treatments.
Early research on the propagation of epithelial keratinocyte cells indicated that disaggregated epidermal cells only grew in monolayers to a very limited extent, and could not be satisfactorily subcultured. In a significant early study, clonal growth of human epidermal keratinocytes was obtained by plating human skin cells with a semi-confluent feeder layer of lethally irradiated 3T3 fibroblast cells which prevented fibroblast overgrowth and promoted multiplication of human keratinocytes in Delbecco's modified medium (DME) supplemented with hydrocortisone (HC) and 20% whole fetal bovine serum (wFBS). The human diploid keratinocytes grown in this system had a finite culture lifetime and low plating efficiency in primary culture (0.1 to 1.0%). On subculture, the plating efficiency rose only occasionally to 10% but was most often in the range of 1 to 5%. Only a range of two to six serial transfers were possible in this system; and, in the absence of 3T3 cells, the human keratinocytes could not even initiate colony formation. [See, Rheinwald, J. G., et al., Cell, 6, pp. 331-334 (1975)].
It was noted in a later study that keratinocytes grown in the lethally irradiated 3T3 cell system were enhanced by the presence of epidermal growth factor (EGF) in the medium. [Rheinwald, J. G., et al., Nature, 265, pp. 421-424 (1977)].
Further definition of an optimal medium for growth of human keratinocytes revealed that only the primary culture of keratinocytes required the presence of the 3T3 fibroblast cells [Peehl, D. M., et al., In Vitro, Vol. 16(6), pp. 516-525 (1980)]. Following the establishment of primary human keratinocyte cultures by the Rheinwald, et al., 3T3 feeder layer method described above, the 3T3 cells were removed on day 3 of culture with ethylene diamine tetraacetic acid (EDTA). Various commercially available test media, each supplemented with hydrocortisone and whole fetal bovine serum and conditioned for 24 hours by irradiated 3T3cells, were added to cultures. Substantial multiplication of human keratinocytes occurred only in conditioned Medium 199, described in Morgan, J. F., et al., Proceedings in the Society of Experimental Biological Medicine, 73, pages 1-8 (1950), in which the stratified keratinocyte colonies grew to confluency and could be subcultured. When cells from the primary culture were inoculated in unconditioned Medium 199, supplemented with an increased concentration of hydrocortisone, whole fetal bovine serum, and pituitary extract fractions, similar growth was achieved, indicating that human keratinocytes, after the primary culture was established, did not require special conditioning factors from fibroblasts for clonal growth and differentiation in culture.
Subsequent studies indicated that commercially available medium F12 eliminated the need for pituitary extract and allowed dialyzed fetal bovine serum protein to be used in place of whole serum for clonal growth media. Adjustments of the composition of medium F12 for optimal clonal growth resulted in a new medium, MCDB151, which supported clonal growth of human keratinocytes with hydrocortisone and fetal bovine serum protein. Optimal growth of human keratinocytes occurred at a very low concentration of calcium ion (0.03 mM) which causes the colonies to remain as monolayers, rather than stratifying as they do in the presence of higher levels of calcium. While medium MCDB151 supported clonal growth of human epidermal keratinocytes with 1.0 mg/ml of fetal bovine serum protein as the only macromolecular supplement, the Rheinwald, et al., feeder layer technique was required for establishment of primary cultures, and subcultures showed a low colony-forming efficiency [See, Peehl, D. M., et. al., In Vitro, Vol. 16(6), pp. 526-538 (1980)].
To develop a medium containing no deliberately added undefined supplements and capable of supporting colony formation of normal human epidermal keratinocytes, hormone and growth factor replacement in the medium was studied [Tsao, M. C., et al., J. Cell. Physiol., 110, pp. 219-229 (1982)]. A new basal medium, MCDB152, was formulated by addition of the trace element supplement from medium MCDB104 [McKeechan, W. L., et al., In Vitro, 13, pages 399-416 (1977)] to medium MCDB151. The ingredients of basal medium MCDB152 therefore included the following: arginine, 1.0.times.10.sup.-3 M; cysteine, 2.4.times.10.sup.-4 M; glutamine, 6.0.times.10.sup.-3 M; histidine, 8.0.times.10.sup.-5 M; isoleucine, 1.5.times.10.sup.-5 M; leucine, 5.0.times.10.sup.-4 M; lysine, 1.0.times.10.sup.-4 M; methionine, 3.0.times.10.sup.-5 M; phenylalanine, 3.0.times.10.sup.-5 M; threonine, 1.0.times.10.sup.-4 M; tryptophan, 1.5.times.10.sup.-5 M; tyrosine, 1.5.times.10.sup.-5 M; valine, 3.0.times. 10.sup.-4 M; alanine, 1.0.times.10.sup.-4 M; asparagine, 1.0.times.10.sup.-4 M; aspartate, 3.0.times.10.sup.-5 M; glutamate, 1.0.times.10.sup.-4 M; glycine, 1.0.times.10.sup.-4 M; proline, 3.0.times.10.sup.-4 M; serine, 6.0.times.10.sup.-4 M; biotin, 6.0.times.10.sup.-8 M; folate, 1.8.times.10.sup.-6 M; lipoate, 1.0.times.10.sup.-6 M; niacinamide, 3.0.times.10.sup.-7 M; pantothenate, 1.0.times.10.sup.-6 M; pyridoxine, 3.0.times.10.sup.-7 M; riboflavin, 1.0.times.10.sup.-7 M; thiamin, 1.0.times.10.sup.-6 M; Vitamin B.sub.12, 3.0.times.10.sup.-7 M; adenine, 1.8.times.10.sup.-4 M; thymidine, 3.0.times.10.sup.-6 M; acetate, 3.7.times.10.sup.-3 M; choline, 1.0.times.10.sup.-4 M; glucose, 6.0.times.10.sup.-3 M; i-inositol, 1.0.times.10.sup.-4 M; putrescine, 1.0.times.10.sup.-6 M; pyruvate, 5.0.times.10.sup.-4 M; calcium, 3.0.times.10.sup.-5 M; magnsium, 6.0.times.10.sup.-4 M; potassium, 1.5.times.10.sup.-3 M; sodium, 1.5.times. 10.sup.-1 M; chloride, 1.3.times.10.sup.-1 M; phosphate, 2.0.times.10.sup.-3 M; sulfate, 4.5.times.10.sup.-6 M; copper, 1.1.times.10.sup.-8 M; iron, 1.5.times.10.sup.-6 M; zinc, 3.5.times.10.sup.-6 M; bicarbonate, 1.4.times.10.sup.-2 M; carbon dioxide, 5%; HEPES, 2.8.times.10.sup.-2 M; phenol red, 3.3.times.10.sup.-6 M; manganese, 1.0.times.10.sup.-9 M; molybdenum, 1.0.times.10.sup.-9 M; nickel, 5.0.times.10.sup.-10 M; selenium, 3.0.times.10.sup.-8 M; silicon, 5.0.times.10.sup.-7 M; tin, 5.0.times.10.sup.-10 M; and vanadium, 5.0.times.10.sup.-9 M. This new basal medium was further supplemented to create the defined medium for growth of human keratinocytes, with epidermal growth factor, 5 ng/ml; transferin, 10 .mu.g/ml; insulin, 5 .mu.g/ml; hydrocortisone, 1.4.times.10.sup.-6 M; ethanolamine, 1.0.times.10.sup.-5 M; phosphoethanolamine, 1.0.times.10.sup.-5 M; and progesterone, 2.0.times.10.sup.-9 M.
In this study, the Rheinwald, et al. feeder layer technique was employed to establish primary cultures, and to suppress overgrowth of fibroblasts and enhance growth of keratinocytes. Clonal growth experiments were performed in the supplemented medium MCDB152. Previous requirements for dialyzed serum and bovine pituitary extract were replaced with the mixture of supplements previously identified. The replacement of whole bovine pituitary extract with ethanolamine and phosphoethanolamine in this study removed the last deliberately added undefined supplement. These researchers were able to contain clonal growth of human epidermal keratinocytes in a chemically defined medium with a low ratio (1:2) of iron concentration (1.5.times.10.sup.-6 M) to zinc concentration (3.times.10.sup.-6 M), with 10 .mu.g/ml transferrin.
Following the initiation of keratinocyte growth in the primary culture, cells in this study were transferred to MCDB152 with supplements. Despite the absence of undefined elements in this medium, several problems were revealed during the course of experimentation. Variable growth of keratinocytes were achieved in MCDB152, but the researchers could not transfer the culture to a third vessel and obtain a viable culture. After the first subculture, the Tsao, et al. researchers achieved a very small colony-forming efficiency (less than 2%). Further, good growth was only obtained in the defined media when the cellular inoculum was prepared from a primary culture using the Rheinwald, et al. technique of 3T3 feeder cells. The cells could not be frozen for storage and subsequently recovered as viable culture-producing cells.
In an alternative attempt to overcome the technical limitations inherent in the Rheinwald, et al. 3T3 feeder layer system for keratinocyte culture, a culture system was developed using human fibronectin. Keratinocytes were established in a primary culture in Delbecco's Modified Eagle's Medium supplemented with 20% fetal bovine serum, hydrocortisone, penicillin G, and streptomycin. The epithelial cells were plated on fibronectin coated plates, requiring ten-fold larger plating densities tha the Rheinwald, et al. technique to obtain comparable colony formation. In this sytem, fibroblast-like cells were apparent in many plates but overgrowth did not occur [See, Gilchrest, B. A., et al., Cell Biology International Reports, Vol. 4, No. 11, pp. 1009-1016 (1980)].
In later related studies human keratinocytes plated on fibronectin-coated cell culture dishes were grown in a cell culture medium consisting of medium 199 with epidermal growth factor, tri-iodothyronine, hydrocortisone, Cohn Fraction IV, insulin, transferin, bovine brain extract, and trace elements. The researchers determined that brain extract was necessary to preserve normal keratinocyte morphology and protein production in this system. [See, Maciag, T., et al., Science, 211, pp. 1452-1454 (1981); Gilchrest, et al., J. Cellular Physiology, 112, pp. 197-206 (1982)]. The Maciag/Gilchrest culture system, characterized by the presence of undefined macromolecular ingredients, i.e., brain extract, Cohn fraction IV and serum, shows no advantage over the Rheinwald, et al. system due to requirements for very high inoculation densities and fibronectin-coated culture vessels in order to achieve an increase in cell numbers per unit time.
From the above description of the state of the art, it will be apparent that there continues to exist a need in the art for methods and materials for securing serum-free production of human epidermal keratinocyte cells. The attempts described thus far have enabled growth of keratinocyte cells from the primary inoculum, but rely on serum components introduced by the feeder layer technique or the use of bovine serum protein in culture medium.
There thus remains a need in the art for materials and methods to provide (1) a source of human epidermal keratinocyte cells for biological research and medical applications grown in serum-free conditions and having a high colony-forming efficiency in serial subculture; (2) a source of human keratinocytes which has no exposure to serum, thereby eliminating the possibility for inducing inflammatory response when used in medical applications; and (3) a source of human keratinocytes having a velocity or efficiency of growth for use in skin grafts thereby contributing to reduced trauma and infection in a patient during the period of exposure of the injury to the environment.